The novel coronavirus disease (also known as COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been swept across the world and emerged as a major health concern [
1] in the recent three years. According to the World Health Organization (WHO), 769,806,130 confirmed cases of COVID-19 have been reported, including 6,955,497 deaths [
2]. The fatality rate has varied significantly by region and age groups. Despite commencing vaccination with initially satisfactory efficacy, the SARS-CoV-2 infection has been going on because of many emergences of variants strains, though it has been declared to be a non-concerning disease now. For example, the Omicron variants have caused global concern owing to their great increased transmissibility and immune evasion capability despite its lesser pathogenicity. Therefore, confirmatory polymerase chain reaction tests to identify the SARS-CoV-2 infection and basic preventive measures, such as social distancing and wearing a mask, remain important precautions against COVID-19 [
3]. The emergence of the Omicron variants has highlighted the need for more alternative therapies with various modes to reduce the impact of mutated strains such as inhibiting SARS-CoV-2 entry/fusion, RNA replication and protease inhibitors, vaccines and drug-free therapies like plasma therapy, etc., and proposed lifestyle factors (such as nitrate-rich and other natural product diets and exercise) as preventive strategies [
4,
5].
2. SARS-CoV-2 and COVID-19
SARS-CoV-2 is a member of the coronaviruses (CoV) family, which are enveloped and single-stranded positive-sense RNA viruses with (typically) a genome of ~30 kilobase (kb). Coronaviruses also have been named after the protruding coronary spikes on the virus’s surface [
9]. Similar with other β-coronaviruses genome organization, SARS-CoV-2 consists of un-translated regions(UTRs) at both 5′ and 3′ end regions and fourteen functional open reading frames (ORFs) that encode for different structural proteins, non-structural proteins (nsps), and accessory proteins [
10,
11]. There are four structural proteins: the spike (S) protein encoded by the S gene is the site on the virus’s surface responsible for binding to the host receptor; the M protein encoded by the M gene shapes the virions and directs envelop formation and provides the matrix for nucleocapsid attaching and budding; the E protein encoded by the E gene is involved in the virus’s assembly and release, contributing to the pathogenesis; and the N gene encodes the N protein, which binds to the RNA genome to maintain the virus’s stability. The ORF1a and ORF1b encode sixteen highly conserved nuclear shuttle proteins (nsp1-nsp16) that are essential for viral replication and transcription processes. Nine accessory proteins provide a selective advantage in the infected host [
12,
13,
14].
Apart from SARS-CoV-2, the other two coronaviruses were found to transmit to human populations, trigger acute respiratory syndromes in humans, and even cause severe outbreak clusters after overcoming the species barrier over the recent two decades, namely the severe acute respiratory syndrome (SARS-CoV-1) in 2002 and the Middle East respiratory syndrome (MERS-CoV) in 2012 [
15,
16]. SARS-CoV-2 shares 80% and 50% similarity with SARS-CoV-1 and MERS-CoV, respectively [
17]. All of them belong to the β-coronavirus genera in the coronaviruses family which infects the respiratory tract, causing atypical pneumonia, and also affects the function of other organs like the liver, heart, kidney, gastrointestinal system, and central nervous system [
18].
COVID-19 is either asymptomatic or mild, with the most common symptoms being fever, headache, dry cough, shortness of breath, and myalgia in about 80–90% of cases, and only around 10% of the infected patients have severe infection with dyspnea, hypoxemia, and extensive radiological involvement of the lung parenchyma. In some critical cases (less than 5%), this virus is likely to cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS), sepsis, and subsequent multi-organ failure leading to respiratory failure and eventually death, which are very similar to the pathological features of SARS and MERS [
19,
20]. Thus, the symptoms vary from individual to individual, ranging from an asymptomatic infection to severe respiratory failure. Gastrointestinal disorders, such as diarrhea, nausea, and vomiting, are reported to a lesser extent. Some patients have also experienced loss of smell and nasal obstruction. This indicated a potential neurotropism of SASR-CoV-2 that may invade the central nervous system [
21]. The individuals with pre-existing comorbidities like obesity, hypertension, diabetes, chronic obstructive pulmonary disease (COPD), cardiovascular disease, cerebrovascular disease, and autoimmune disease or immunosuppressed condition are at a much higher risk of severe COVID-19 disease [
22].
Although SARS-CoV-2 mainly infects bronchial ciliated epithelium and pulmonary type II cells initially, electron imaging has detected leftover virus particles in endothelial cells. SARS-CoV-2’s entry into host cells is mediated by binding to the host cellular receptor, angiotensin-converting enzyme 2 (ACE2), which is located on the host cell surface of the target organs, with a higher affinity than with the one measured with SARS-CoV-1. ACE2s are highly expressed in type II alveoli epithelial cells, which serve as the primary targets for viral attacks [
23]. Studies identified that the virus significantly impacts other organs with the development of myocarditis, gastrointestinal disturbances, renal ailments, and irregular blood pressure, and the presence of ACE2 on the epithelial and endothelial lining of the liver, heart, kidney, pancreas, gastrointestinal tract, genital organs, thyroid, blood vessels, and so on is considered partly responsible for this [
24]. In the case of SARS-CoV-2, S protein, which is required for viral entry, has two regions, S1 and S2. S1 has a receptor-binding domain (RBD) that mediates direct contact with ACE2 to form the S protein RBD-ACE2 complex, whereas S2 is involved in subsequent membrane fusion [
25].
After SARS-CoV-2 enters the target cells, the virus is disassembled to release viral RNA into the cytoplasm for translation of non-structural proteins and structural proteins and replication of genome. The translated replicase components rearrange the endoplasmic reticulum (ER) into double-membrane vesicles (DMVs) that facilitate viral replication of genomic and subgenomic RNAs. The latter are translated into accessory and viral structural proteins to facilitate virus particle formation. The virus particles germinated in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) were exocytosed into the extracellular compartment for the propagation of the infection in other target cells [
29].
In parallel with the ongoing viral replication, the viral RNA genome release into the cytoplasm is detected by intracellular pattern recognition receptors (PRRs), innate immune sensors such as the endosomal Toll-like receptor, and cytosolic retinoic acid-inducible gene I-like receptors. Following PRR activation, molecular signaling cascades culminate in the activation of downstream transcription factors, such as nuclear factor-κB (NF-κB), to produce numerous pro-inflammatory cytokines (including interferon (IFN)-α, IFN-γ, interleukin (IL)-1β, IL-6, IL-12, IL-18, IL-33, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β) and chemokines (such as chemokine ligand (CCL) 2, CCL3, CCL5, CXC chemokine ligand (CXCL) 8, CXCL9, and CXCL10) in the form of a “cytokine storm” [
29,
32]. Multiple clinical symptoms are strongly connected with the release of these cytokines.
Another pathway pertaining to the pathogenesis of COVID-19 is the ACE2-angiotensin 1-7 (Ang 1-7) axis. It is known that ACE converts angiotensin I (Ang I) into the pro-inflammatory peptide angiotensin II (Ang II), and ACE2 metabolizes Ang II to produce Ang 1-7. The SARS-CoV-2 infection downregulates ACE2 expression by internalizing it with viral particles from the host cell surface and fails to catalyze the conversion of Ang II to Ang 1-7, resulting in Ang II accumulation. A high concentration of Ang II can cause increased inflammatory responses and reactive oxygen species (ROS) [
37,
38]. A large number of activated pro-inflammatory cytokines and chemokines were found in the serum of patients with severe COVID-19, including the membrane forms of epidermal growth factor (EGF) family members, IL-6 receptor, and TNF-α, and developed into a strong cytokine storm [
39]. When high inflammation persists for a long time, it damages many tissues and organs and contributes to an imbalance of ROS, which leads to vasoconstriction.
3. Role of NO in SARS-CoV-2 Infection
3.1. Antiviral Effect
From the early days, it was warranted that NO has a rather broad spectrum of antiviral effects and inhibits viral replication, including ectromelia virus, vaccinia, vesicular stomatitis virus, adenovirus, murine CMV (MCMV), murine retrovirus, rhinovirus, herpes simplex-1 viruses, HIV, hantavirus, influenza, Japanese encephalitis virus, and (most importantly) coronavirus [
42,
43,
44,
45,
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63]. More notably, NO has been shown to impair SARS-CoV and SARS-CoV-2 replication in light of the COVID-19 pandemic because of their similar infection processes [
30,
31,
64,
65]. Two NO-mediated antiviral mechanisms were proposed, and were later experimentally verified, as follows:
- (1)
-
NO decreased the palmitoylation level of S protein, thereby interfering with binding to the target receptor on the host cell. Three different studies demonstrated the potential of NO compounds in the inhibition of SARS-CoV replication in a concentration-dependent manner [
30,
64,
65], and the effect of NO on S protein was also investigated. The results showed that the NO donor-S-nitroso-N-acetylpenicillamine (SNAP) treatment significantly reduced the number of palmitoylated S protein, and the intercellular fusion was significantly decreased. Also, the entry efficiency of the pseudo-type virus was significantly lower after SNAP treatment, and the virus infection rate decreased by about 70% [
30].
- (2)
-
NO affected replication-related cysteine proteases which directly inhibited viral RNA replication. Similar to the Coxsackievirus 3C cysteine protease, SARS-CoV-2 3CL cysteine protease may be a potential target for S-nitrosation, causing a suppression of the protease activity and a resultant decrease in viral replication [
56]. The in vitro study by Akaberi et al. showed that SARS-CoV-2 3CL recombinant protease was covalently inhibited by SNAP through the transfer of nitrosonium ions (NO
+s) to the protease cysteine residue, and the observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosylation of the enzyme active site cysteine. Although the viral replication was not completely abolished, SNAP delayed or completely prevented the development of the viral cytopathic effect in treated cells [
31]. In addition, the analysis of proteolytic degradation of the viral polypeptide showed that the content of the nucleocapsid N protein was drastically decreased in the presence of SNAP and that high-molar-mass (non-processed) polypeptide content was increased [
30,
65].
3.2. Effect on Inflammation
Inflammation is a critical defensive mechanism for inactivating pathogens, removing irritants, and paving the way for tissue repairs. However, excessive inflammation causes injury. Studies have shown that NO, as a ubiquitous signaling molecule, plays a role in almost every stage of inflammation [
67].
For example, NO suppresses the production of a large number of cytokines in lymphocytes, eosinophils, monocytes, and other immune cells, including key cytokines in the inflammatory response [
54,
68,
69]. The different and inappropriate inflammatory response associated with the SARS-CoV-2 infection in the context of the COVID-19 illness was described above (SARS-CoV-2 and COVID-19 section). Moreover, multiple reports indicated that NF-κB is involved in the upregulation of inflammatory responses in patients with the SARS-CoV-2 infection as a potential main regulator of this process [
70]. Regulation of NF-κB activation by NO has been well investigated for its involvement in various physiological and pathological conditions [
71]. The most common form of active NF-κB is a heterodimer consisting of the protein subunits p50 and p65. After the IκB-kinase (IKK) complex phosphorylates the cytoplasmic inhibitor factor IκB, which normally sequesters NF-κB in an inactive form in the cytosol, NF-κB is translocated into the nucleus and induces a plethora of pro-inflammatory gene expressions. Here, NO represses IκB-kinase through S-nitrosylation and S-nitrosylation of IKKβ at Cys179, and p50 at Cys62 inhibits NF-κB-dependent DNA binding, promoter activity, and gene transcription, leading to the subsequent inflammatory response [
72].
An additional factor that contributes to the excessive inflammatory responses is relevant to ACE2. ACE and ACE2 serve opposing physiological functions. After ACE cleaves Ang I to Ang II, Ang II binds its receptor to constrict blood vessels. In addition, ACE inhibits NO production, promoting ROS and inflammation. An excess of ROS damages endothelial dysfunction, permeable vessels, and lipid membrane peroxidation [75]. On the contrary, ACE2 inactivates Ang II and generates Ang 1-7, which promote endothelial production of NO, as both potent vasodilators and inhibitors of ACE. The accumulation of Ang II caused by the downregulation of ACE2 expression in the SARS-CoV-2 infection can induce vasoconstriction and act as a pro-inflammatory cytokine via AT1R. The AngII-AT1R axis induces inflammatory cytokines, including TNFα and IL-6-soluble (s)IL-6R, via activating disintegrin and metalloprotease 17 (ADAM17), followed by the activation of the IL-6 amplifier (IL-6 AMP), which describes enhanced NF-κB activation machinery via the coactivation of NF-κB and the signal transducer and activator of transcription-3 (STAT3) [39].
Inflammation-induced platelet activation, which can lead to increased coagulation and consequent diseases, can be lessened by NO [78]. NO maintains physiological vascular homeostasis in tissues and protects blood vessels from damage with platelets and circulating cells, and the decrease in endothelial NO production is a sign of endothelial dysfunction and thrombotic events [79]. The decreased or ceased release of NO following endothelial cell dysfunction leads to the accumulation of free Ca2+ in vascular smooth muscle cells, continuous vasoconstriction, and subsequently a blood hypercoagulable state. When blood vessels are damaged, platelets quickly gather to the injured site to form platelet clots and a complex with plasma factor VIIa, whose subsequent interaction with extravascular tissue factor initiates the action of thrombin (via conversion of inactive protease factor X into the active protease factor Xa). Thrombin then converts soluble fibrin into insoluble fibrin, which makes the platelet clot entangled with blood cells to form a thrombus.
3.3. Effects on Vasodilation
NO can serve as an effective vasodilator regulator. It effectively relaxes smooth muscle cells and dilates blood vessels to improve oxygenation and reduce pulmonary vascular resistance and promoter oxygen inhalation, thus increasing the blood flow of capillaries, increasing the exchange gas with alveoli, and accelerating oxygen circulation in the body, which may improve respiratory symptoms.
In detail, NO reacts with oxygen to form nitrogen dioxide and nitrite, resulting in pulmonary vasodilatation. Further, nitrosylation of the cysteine residue of the haemoglobin β subunit leads to the formation of a stable derivative that retains vasodilatory properties which can increase blood flow and oxygen delivery to the system’s vasculature [
82]. NO regulates the vascular tone via the cyclic guanosine monophosphate (cGMP)-dependent mechanism. It binds to soluble guanylate cyclase (sGC) and activates it, resulting in the production of intracellular cyclic guanosine monophosphate (cGMP). cGMP reduces the intracellular Ca
2+ concentration and relaxes smooth muscle cells. The reduction in calcium reduces the ability of myosin light-chain kinase (MLCK) to phosphorylate the myosin molecule, preventing cross-bridging and thus enhancing the relaxation of smooth muscle cells and promoting blood flow. It also activates potassium channels, leading to hyperpolarization and relaxation [
83].
4. Application of NO in Clinical Treatment of COVID-19
Based on the aforementioned antiviral, anti-inflammatory, and anti-thrombotic effects, NO, also as a potent and selective pulmonary vasodilator, has been appraised as an attractive agent that may be beneficial to COVID-19 patients’ therapy, with or without ARDS [6,7]. Further, NO inhalation therapy showed promising potency in the 2003 SARS outbreak [88]. Many case series, cohort studies, retrospective investigations, and clinical trials that investigated different strategies of NO administration under various conditions were conducted and analyzed to discuss the use of exogenous NO therapy among patients with COVID-19.
Many clinical observations and studies have demonstrated that iNO treatment produced an acute improvement in the systemic oxygenation process in hypoxemic patients and prevented the progression of hypoxemic respiratory failure [103,110]. High-dose NO (160–200 ppm for 30 min) was safely administered to pregnant females with severe COVID-19 pneumonia and was associated with improved oxygenation, respiratory rate, and cardiopulmonary system function and a decrease in systemic inflammation [93]. Another strategy for iNO administration in COVID-19 is the administration of a long-term, constant NO insufflation at low doses, which may increase antiviral activity (dose and time-dependent) and reduce the severity of the disease and time to recovery in patients with COVID-19 [122,123]. Intriguingly, a randomized clinical trial using iNO among healthcare workers was conducted to prevent them from being infected with the SARS-CoV-2 during their work. The use of iNO in conjunction with pharmaceutical vasodilators, such as almitrine and prostaglandin, has also shown a positive clinical value as a rescue therapy to enhance oxygen levels in patients with COVID-19 [98,115,119].
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